Ihara Hideshi, Himeno Michio
Department of Biological Science, Graduate School of Science, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan.
J Invertebr Pathol. 2008 Jun;98(2):177-83. doi: 10.1016/j.jip.2008.02.007. Epub 2008 Feb 21.
The binding of Bacillus thuringiensis delta-endotoxin to brush border membrane vesicles (BBMVs) from the target insect larval midgut comprises with not only a reversible but also an irreversible component. The irreversible binding of delta-endotoxin is thought to be a pathologically important factor. Here, we studied the irreversible binding of Cry1Aa to the BBMVs of Bombyx mori. The (125)I-labeled Cry1Aa bound to the solubilized brush border membrane (BBM) through rapid dissociation only, unlike the binding to BBMVs, indicating that the toxin bound to the solubilized BBM through only a reversible process. Low-temperature sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that the toxin bound irreversibly to BBMVs formed an oligomer of 220kDa, whereas that bound reversibly to the solubilized BBM did not oligomeraize. When the (125)I-labeled Cry1Aa bound irreversibly to the BBMVs was digested by proteinase K, approximately 40% of the toxin observed to be resistant to proteinase K. The molecular mass of the toxin resistant to proteinase K was 60kDa, suggesting that the irreversible binding comprise two forms. These results support the notion that the irreversible binding of the toxin to BBMVs is due to the insertion of the toxin into the lipid bilayers and oligomerization to form channels.
苏云金芽孢杆菌δ-内毒素与目标昆虫幼虫中肠刷状缘膜囊泡（BBMVs）的结合不仅包含可逆成分，还包含不可逆成分。δ-内毒素的不可逆结合被认为是一个具有重要病理意义的因素。在此，我们研究了Cry1Aa与家蚕BBMVs的不可逆结合。与结合到BBMVs不同，（125）I标记的Cry1Aa仅通过快速解离与溶解的刷状缘膜（BBM）结合，这表明毒素仅通过可逆过程与溶解的BBM结合。低温十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示，不可逆结合到BBMVs的毒素形成了一个220kDa的寡聚体，而可逆结合到溶解的BBM的毒素没有寡聚化。当不可逆结合到BBMVs的（125）I标记的Cry1Aa被蛋白酶K消化时，约40%的毒素对蛋白酶K具有抗性。对蛋白酶K具有抗性的毒素的分子量为60kDa，这表明不可逆结合包括两种形式。这些结果支持了毒素与BBMVs的不可逆结合是由于毒素插入脂质双层并寡聚化形成通道这一观点。
